Abstract

The pH-sensing chaperone HdeA promotes the survival of enteropathogenic bacteria during transit through the harshly acidic environment of the mammalian stomach. At low pH, HdeA transitions from an inactive, folded, dimer to chaperone-active, disordered, monomers to protect against the acid-induced aggregation of periplasmic proteins. Toward achieving a detailed mechanistic understanding of the pH response of HdeA, we develop a multiscale modeling approach to capture its pH-dependent thermodynamics. Our approach combines pK(a) (logarithmic acid dissociation constant) calculations from all-atom constant pH molecular dynamics simulations with coarse-grained modeling and yields new, atomic-level, insights into HdeA chaperone function that can be directly tested by experiment. "pH triggers" that significantly destabilize the dimer are each located near the N-terminus of a helix, suggesting that their neutralization at low pH destabilizes the helix macrodipole as a mechanism of monomer disordering. Moreover, we observe a non-monotonic change in the pH-dependent stability of HdeA, with maximal stability of the dimer near pH5. This affect is attributed to the protonation Glu37, which exhibits an anomalously high pK(a) value and is located within the hydrophobic dimer interface. Finally, the pH-dependent binding pathway of HdeA comprises a partially unfolded, dimeric intermediate that becomes increasingly stable relative to the native dimer at lower pH values and displays key structural features for chaperone-substrate interaction. We anticipate that the insights from our model will help inform ongoing NMR and biochemical investigations.

Folded dimer and unfolded monomer structures of HdeA. (a) Crystal structure of the HdeA homodimer (PDB ID: 1BG8). The acidic residues that exhibit the most significant destabilizing (Asp20, red) and stabilizing (Glu37, pink) effects on HdeA upon protonation at low pH are denoted as van der Waals spheres. (b–d) Representative conformers of the unfolded ensemble of the HdeA monomer from clustering of the coarse-grained ensemble. The corresponding clusters have the following fractional population sizes: (b) 0.24 (c) 0.41, and (d) 0.35. The N-terminal residue in each unfolded conformer is denoted in CPK representation.

Contribution of acidic residues to the pH-dependent binding stability of HdeA. In the binding stability curves, ΔΔG = 0 at the reference pH (pHref = 4). A decrease in ΔΔG from pHref to a given target pH represents destabilization of the dimer complex and thus favors the monomeric state. An increase in ΔΔG from pHref to a target pH stabilizes binding in the complex and thus favors the dimeric state. The black curve in the upper left corresponds to the binding stability profile with the contributions from all acidic residues in the dimer. Red and cyan curves denote acidic residues that destabilize and stabilize the dimer upon protonation at low pH, respectively. In the bottom right is a cartoon representation of the HdeA dimer crystal structure. In the structure, lines are drawn between residue pairs that are in close contact in the crystal structure and that involve an acidic amino acid. Red and cyan lines denote contacts involving acidic residues that destabilize and stabilize, respectively, the dimer at low pH. The location of the acidic residues that exhibit the largest destabilizing and stabilizing contributions are denoted by red (Asp20, Asp25, and Asp51) and cyan (Glu37) beads, respectively. For clarity, labels for these residues are only shown in one of the two monomers. Asp2, Asp8, and Asp88 are not resolved in the HdeA crystal structure, and thus were not considered in our calculations.

pH-dependent binding pathway in HdeA. (a) Free energy profiles as a function of Qtotal at different pH values. The basins corresponding to the native dimer (N2), partially unfolded dimeric intermediate (I2), and unfolded monomers (2M) are indicated above their respective basins. (b) The free energy projected onto the plane of Qtotal and the distance between the monomer centers of mass (dCM). The conformational basins are labeled on the pH 7 surface and follow the same abbreviations as in (a). The colorbars indicate the free energy in units of kcal/mol computed at 0.98 Tm.

Proposed key structural features for HdeA chaperone activity. (a) Representative conformation of the partially unfolded, dimeric intermediate (I2). Hydrophobic residues (white), lysines (cyan), glutamates (pink), and aspartates (red) are indicated on the cartoon backbone. The side chain of Trp16, which becomes exposed upon the formation of I2, is highlighted. (b) HdeA dimer with Glu37 (pink van der Waals spheres) depicted in the context of hydrophobic residues (grey surface). All other acidic residues are displayed in stick representation (Glu residues in pink and Asp residues in red). In both panels, the labeled monomer is colored black and the neighboring subunit is shown in yellow.